The quality control method and standard were established to control the quality of Pteris multifida in this paper. The tests of water content, total ash, acid-unsoluble ash and ethanol-soluble extractives of P. multifida were carried out according to the methods recorded in appendix of Chinese Pharmacopeia (2010 edition, volume 1) . The TLC method was established by using rhoifolin as references, and a mixture of CHCl3 -MeOH-HAc (6: 1: 1) as the developing solvent system on GF254 thin layer plate. The contents of rhoifolin was determined by HPLC on a Diamonsil C18 (4.6 mm x 250 mm, 5 μm) column, using acetonitrile-water (containing 0.15% formic acid) (16: 84) as mobile phase at a flow rate of 1.0 mL x min(-1). The column temperature was 30 degrees C and the detection wave-length was 350 nm. As a result, pterosin C 3-O-β-D-glucosidede and the other constituents were well separated on TLC detected under the UV light at 254 nm . The methodology validation for the assay of rhoifolin presented that it was in good linear correlation in the ranges of 0.025 5-5.1 μg with the regression equations of Y = 1 092.4X + 9.503 5 (r = 0.999 8), and the average recoveries were 100.3% (RSD 1.3%). The content range of rhoifolin from 16 different batches of Pteris multifida was 0.08-5.06 mg x g(-1). The water content, total ash, acid-unsoluble ash and ethanol-soluble extractives of 16 samples varied in the ranges of 7.35% - 12.96%, 6.90% - 16.33%, 2.07% -11.38% and 13.29% -23.87%, respectively. The suggesting limes in the quality standard for water content, total ash, acid-unsoluble ash, ethanol-soluble extractives and rhoifolin content were ≤ 12% , ≤ 15% , ≤ 8.5% , ≥ 14% and ≥ 0.040%, respectively. The result proved that the established quality of control method was specific and accurate, which can be used for the quality control of P. multifida.
China ; Chromatography, High Pressure Liquid ; Chromatography, Thin Layer ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; standards ; Pteris ; chemistry ; Quality Control

Objective To construct lentiviral vector miR30-CD133 targeting CD133 gene and investigate its effects on SMMC7721 cells. Methods Thespecific sequence of DNA which containing both sense and antisense Oligo DNA of the targeting sequence were designed, synthesized and cloned into the pPRIME vector. pPRIME-miR30-CD133, psPAX2 and pMD2G were co-transfected on 293T cells to get the lentivirus the transfection efficiency was investigated by confocal laser scanning microscope. The expression of miR30-CD133 on CD133 were detected by qRT-PCR and Western blott. The proliferation and apoptosis effect of miR30-CD133 was respectively evaluated by CCK-8 assay and AnnexinV/PI. Results Functional PFU titers of PRIME-miR30-CD133 were 6.58 × 109 PFU/mL. The expression of CD133 mRNA in sh CD133 group (0.18 ± 0.04) was less than Blank group and shNC group (P<0.05). The expression of CD133 protein in sh CD133 group was less than Blank group (10%) and shNC group (8%) (P < 0.05). Cells proliferation activity were significantly inhibited in shCD133 group compared with control group. Apoptosis rate were significantly increased in shCD133 group (41.3%) compared with Blank group (25.3%)and shNC group (24.3%). Conclusion The lentivirus miR30 vector targeting CD133 gene was successfully constructed, which will be a basis to the further CD133 gene studies.

Objective To isolate and culture the rabbit mesenchymal stem cells,and explore the biological features in vitro.Methods Two milliliter of bone marrow was extracted from the tibia of juvenile rabbit under anesthesia and sterile conditions,and mesenchymal stem cells were obtained by density gradient centrifugation and inoculated to 100ml culture flask.The morphological changes,adherence rate and ultrastructure of the stem cells were observed.The surface markers and growth cycle of the mesenchymal stem cells were detected by flow cytometry,and biological features were observed by inducing the cells differentiate into osteoblasts.Results The adherence of mesenchymal stem cells started 2 hours after culturing,basically finished 20 hours later,and more than 90% of the cells were fused at the 15th day.Observed with transmission electron microscopy,the cultured cells showed typical ultrastructure of stem cells: numbers of microvillus mainly distributed on one side of the cells,irregular nuclei with indentation,and lots of cell organs.Analysis for the cell surface markers indicated positive expression of CD44 and CD90 and negative expression of CD45 in mesenchymal stem cells.Cell growth cycle assay showed that most mesenchymal stem cells(about 97%) stayed in stage G1 and G2.Three weeks after osteoblastic induction,the mesenchymal stem cells gradually produced small calcium salt crystals,the cells were positive in alkaline phosphatase staining,and Von Kossa staining showed many calcium salts in the cell clone as black reaction deposits.Conclusions The rabbit bone marrow mesenchymal stem cells isolated by density gradient centrifugation are of high purity and stable growth rate,and the cells' characters can be still maintained after serial subcultivation.Since easy to operate on isolation and proliferation,the mesenchymal stem cells could be used as the tissue engineering seed cells in repairing of intervertebral disc degeneration.

Objective To apply the oral gastrointestinal ultrasound contrast agents for evaluating the gastric motility abnor‐mality in the patients with anxiety disorder .Methods Twenty patients with anxiety disorder complicating upper digestive tract symptoms without organic pathological changes were enrolled as the anxiety disorder group .Twenty healthy volunteers were en‐rolled as the control group in this study .The two groups orally took gastrointestinal ultrasound contrast agents .The antral contrac‐tion frequency ,antral contraction amplitude amd MI were measured at each time point .GER was calculated .The the gastric motility parameters in the patients with anxiety disorder were evaluated .Results The antral contraction frequency and MI at initial 2 min had no statistical difference between the anxiety group and the control group .The antral contraction amplitude ,antral contraction frequency amd MI at each time point during 5 -10 min after contrast in the anxiety disorder group were significantly decreased compared with the control group ,and the differences were statistically significant (P< 0 .05) .After 20 min ,GER in the control group was significantly higher than that in the anxiety group ,the difference was statistically significant (P<0 .05) .Conclusion O‐ral gastrointestinal ultrasound contrast agents is a economic ,strongly operable ,non‐invasive and highly repeatable method for evalu‐ating the gastric motility .

BACKGROUND: Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a kind of cytokine which can stimulate the differentiation and proliferation of haematopoictic precursor cells and plays an important role in the process of immunoloregulation. Escherichia coli (E.coli) expression system has advantages,such as low cost and high output, over other systems.Therefore, E.coli is still the most commonly used exogenous gene expression system.OBJECTIVE: To observe the expression of hGM-CSF in the E.coli swain DH5α.DESIGN, TIME AND SETTING: The present observational experiment was performed at the Central Laboratory of Biotechnology Research,China Three Gorges University between February and June 2007. MATERIALS: Plasmid pBR322hGM-CSF was purchased from American Type Culture Collection, USA.hGM-CSF antibody was sourced from Sigma Company, USA.IPTG, X-gal,and vector pMGT-18 were purchased from Shanghai Bioengineering Technology Company, China. METHODS: Primer was designed according to hGM-CSF gene. Taking cloning vector plasmid pBR322-hGM-CSF as template, hGM-CSF gene was acquired and recombined into prokaryotic expression vector pGEX-4T-1. Recombinant plasmid confirmed by enzyme digestion and sequencing was transformed into E.coli strain DH5α and induced by isopropy-β-D-thiogalactoside (IPTG). Expression product was identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and detected by Western blotting assay. MAIN OUTCOME MEASURES: hGM-CSF expression in the E.coli strain DH5α. RESULTS: SDS-PAGE results revealed that the recombinant hGM-CSF protein with relative molecular weight of 40 500 was produced up to approximately 17.9% of total protein. Western blotting detection results indicated that there was a 40 500 bp brand. CONCLUSION: The present study reconstructed prokaryotic expression vector pGEX-hGM-CSF, induced its expression in the E.coli strain DH5α, and obtained hGM-CSF fusion protein.

AIM: To detect the serum proteomic patterns in patients of breast cancer by the method of SELDI-TOF-MS and CM10 ProteinChip, and to screen the biomarker candidates, build and validate the diagnostic models, and evaluate its clinical value in surveillance and follow-up after operation. METHODS: The SELDI-TOF-MS technology and CM10 ProteinChip were used to detect the proteomic patterns of serum from 63 breast cancer patients and 40 healthy women. The biomarker candidates were screened and the diagnostic models were constructed by ZJU-PDAS software. Meanwhile, the model was blind-validated in another 23 patients and 20 healthy women. At the same time, 16 serum samples were detected to evaluate its value in surveillance and follow-up after operation. RESULTS: The best model was composed by two protein peaks (BC1/3.9 kD and BC2/5.6 kD) with its sensitivity and specificity of 87.30% (55/63) and 95.00% (38/40), respectively. The sensitivity and specificity in the blind-validation of new cases were 95.65% (22/23) and 85.00% (17/20), respectively. The diagnostic efficacies were the same to the patients of different stages (P>0.05). The expression of BC1 increased while BC2 decreased after operation. The expression of BC2 in the patients with recurrence or metastasis was higher than that in the tumor-free survivors (P<0.05). CONCLUSION: This method shows its potential in detection, surveillance and follow-up after operation. The method is also useful for screening the novel and better biomarkers in breast cancer.

Objective:To investigate the existence of side population cells with the potency of stem cells in human gallbladder carcinoma cell line GBC-SD and the differences in ABCG2,Oct-4 and CD34 expression among SP cells,non-SP cells and GBC-SD cells.Methods:SP and non-SP cells were sorted from GBC-SD cells by fluorescence-activated cell sorting(FACS).The expression of ABCG2,Oct-4 and CD34 in SP cells,non-SP cells,and GBC-SD cells was detected by reverse transcription-polymerase chain reaction(RT-PCR),Western blot,flow cytometry(FCM)and immunofluorescence chemistry.Results:SP cells with stem cell potency were isolated from GBC-SD cells with a proportion of(0.64±0.08)%.The metastatic ability of SP cells was obviously higher than that of non-SP cells and GBC-SD cells(P<0.05).The expression of ABCG2 was significantly higher in SP cells than in non-SP cells and GBC-SD cells[(89.56±3.86)%vs.(1.32±0.49)%and(12.37±1.61)%,P=0.001].The expression of Oct-4 in these cells was(94.87±1.40)%,(88.16±2.34)%and(90.17±1.61)%,respectively(P>0.05).CD34 was neady absent in these cells on protein level[(1.78±0.51)%vs.(0.63±0.21)%and(0.96±0.381)%,P>0.05)],but it was highly expressed in non-SP cells and GBC-SD cells and absent in SP cells off mRNA leve;.Conclusion:SP calls which hava the potency of stem cells,exist in human gallbladder carcinoma GBC-SD cell line and have the phenotype of ABCG2+Oct-4+CD34-.

Background and purpose:With the widespread use of screening of prostate-specific antigen (PSA) levels, prostate cancers at organ-conifned stage are increasing in newly diagnosed cases. However, some defects remain in conventional monoexponential diffusion-weighted imaging (DWI) for differentiating organ-conifned prostate cancer from benign lesions. Therefore, the aim of this study was to obtain biexponential apparent diffusion parameters of prostate organ-conifned cancer, chronic prostatitis in peripheral zone (PZ) and normal PZ tissue, and to compare with monoexponential apparent diffusion coeffcient (ADC) for differentiating prostate cancer from prostatitis lesions. Methods:Sixteen patients with pathologically confirmed prostate organ-confined cancer in PZ, 14 with prostatitis underwent conventional (b-factors 0, 1 000 s/mm2) and 10b-factors (0-3 000 s/mm2) diffusion-weighted imaging (DWI).The monoexponential ADC value and biexponential parameters fast ADC (ADCf), fraction of ADCf (f), slow ADC (ADCs) value for prostate cancer, prostatitis and normal tissues were calculated and compared. Receiver operating characteristic analysis was performed for those parameters.Results:Biexponential and monoexponential parameters were obtained for 18 prostate cancers, 18 prostatitis and 37 normal PZ tissues. The ADC value of prostate cancer tissues was remarkably lower [(0.83±0.11)×10-3 mm2/s] than that of other tissues (P<0.01), while the ADC value of prostatitis [(1.45±0.19)×10-3 mm2/s] was lower than that of PZ [(1.67±0.31)×10-3 mm2/s] (P<0.01). Prostate cancer tissues had low-er ADCf [(1.54±0.23)×10-3 mm2/s],f [(45.8±5.4)%] and ADCs [(0.52±0.15)×10-3mm2/s] than the other tissues (P<0.01). The ADCf,f and ADCs were higher in PZ [(3.90±0.40)×10-3, (67.3±8.2)% and (1.51±0.36)×10-3 mm2/s] than prostatitis [(3.06±0.49)×10-3, (47.9±3.9)% and (0.91±0.29)×10-3 mm2/s) (P<0.01). The area under the curve (AUC) of ADCf and ADC were similar in differentiating cancer and prostatitis (0.96vs 0.94) (P>0.01), but the AUC off and ADCs in differ-entiating cancer from prostatitis (0.83 and 0.80) were signiifcantly lower than that of ADC (P<0.01).Conclusion:The biexponential DWI provided additional tissue characterization parameters for different prostate tissues. ADCf yielded comparable accuracy with ADC in identiifcation of prostate organ-conifned cancer. The biexponential parameter could further improve the diagnostic effcacy.

Objective:To explore the stress change rule of craniofacial bone suture and the interface of bone-implant against differ-ent strength and direction of protraction on the implant anchorage in alveolar bone.Methods:The original DICOMdata of 2-D image of craniofacial complex were obtained by high resolution CT scanning.3-D finite element models of craniofacial complex were devel-oped according to the DICOMdata.Forces of 1 -1 0 N inclined at 0 -60°to Frankfort horizontal plane in the anterior and inferior di-rections were respectively applied on the implant anchorage in the alveolar bone at 32 23 .Data of principle stress and Von Mises Stress of each mode of each simulaton was caculated.Results:The change rule of the effectiveness of different force value of protrac-tion in the same direction was the same in different stress zone;that of the same force value of the protraction in differente direction differed in different stress zone.When the protraction angle was less than 30°,the maxillary complex will spin up.In the 30°,the maxillary complex showed the forward growth.Between 40°-50°,the growth direction was the same with the protraction direction. When the protraction angle was more than 50°,the maxillary complex showed down spin.Conclusion:Protraction force of 1 -1 0 N at 30°-50°to Frankfort horizontal plane on implant anchorage in the alveolar bone at 32 23 can induce maxillary complex grow for-ward.

Objective To investigate the influence of age on clinical features and prognosis of severe traumatic brain injury.Methods A total of 135 patients with severe traumatic brain injury were divided into four groups according to age:the juvenile group (＜18 years,15 cases),the young adult group (18 44 years,77 cases),the middle aged group (45 59 years,37 cases) and the elderly group (＞60 years,6 cases).Neurological functions were assessed by the Disability Rating Scale (DRS),the Mini-Mental Status Examination (MMSE),the Fugl-Meyer motor scale (FM) and the Modified Barthel Index (MBI).All the patients were followed up with DRS evaluation 1 4 years after discharge from the hospital.Results MMSE scores decreased with age,with statistically significant differences between the elderly group and the juvenile group,the young adult group and the middle aged group,respectively [(11.0±5.2) vs (21.5±8.1),(21.4±8.0) and (19.1±8.1),respectively; t=2.663、2.825、2.561,P=0.015,0.006,0.022,respectively].Similarly,when compared with the other groups,the elderly group also showed statistically significant differences in follow-up DRS scores [(12.8±6.1) vs.(4.3±3.6),(6.7±5.0) and (7.8±6.9),respectively; t=-2.382、-2.587、-2.385,P =0.040,0.013 and 0.038,respectively]; and the DRS score differentials [(2.3±4.6) vs.(6.2±4.3),(6.7±3.1) and (4.6±3.1); t=2.366、2.242、2.626,P =0.004,0.013 and 0.009,respectively].Conclusions Age may be one of the factors that affect cognitive functions and prognosis associated with traumatic brain injury,and the prognosis for elderly patients is generally unfavorable.